Inhibition or lack of axon guidance affects neuronal cell motility and positional maintenance, resulting in the failure to regenerate neurona! connections after injury. Defective guidance is also associated with diseases ranging from Alzheimers to cancer. The long term objective of our research is to determine the biophysical basis of signal transduction mechanisms involving axon guidance cue receptors and small GTPases. The Plexin-B1 transmembrane protein is the first example of a receptor that interacts directly with small GTPase, Rac1. The major goal of the proposed project is to provide a detailed structural and thermodynamic characterization for the basis of the plexin-Rac1 interaction and to understand its role in plexin mediated signal transduction. Our hypothesis is that the propensity to populate different conformational states is already a characteristic of the Rac1 Binding Domain, and that such regulatory switching behavior will be apparent in this plexin domain by itself, if not upon its interaction with the GTPase or in response to other perturbations. Specific aims: 1. Determination of the structure of the Rac1 binding domain of plexin-B1 and characterization of its dimerization interface and of the putative Rac1 binding switch. Nuclear Magnetic Resonance (NMR) spectroscopy will be used for structure determination of a monomeric form of plexin, and will be utilized to map the plexin dimerization interface of the wild type protein. 2. Characterization of the plexin Rac1 binding domain - GTPase interface, also in a non-binding mutant, and determination of the structure of the protein plexin-Rac1 complex. 3. Thermodynamic perturbations caused by plexin:GTPase complexation will be quantified and the relationship between stability and binding affinity of the structures involved will be determined. Global thermodynamic perturbations will be followed by fluorescence and circular dichroism spectroscopy, site specific changes by amide hydrogen exchange in conjunction with NMR spectroscopy. Significance: These studies will determine the biophysical basis for a signaling mechanism involving a small GTPase - protein interaction and will provide deeper insights into other interactions that involve small GTPases in signaling events.